The granule exocytosis model of lymphocyte-mediated cytotoxicity postulates an antigen-triggered rapid secretion of the preformed potent lytic agent perforin (as well as other mediators) into the synapse-like space between the cytotoxic lymphocyte and its bound target. However, the question of why the effector cell does not itself die of perforin damage has often been raised without a clear answer. We have recently carried out a series of experiments designed to test a new model for self-protection which proposes that cysteine proteases on the effector cell membrane inactivate inserted perforin before it can aggregate and form pores. We have tested this model by asking if 1) active cysteine proteases are detectable on the surface of cytotoxic lymphocytes, and 2) cysteine protease inhibitors block self-protection and result in degranulation-induced death in cytotoxic lymphocytes. We have detected active cysteine proteases on the surface of cytotoxic T lymphocytes by labeling them with a biotinylated cathepsin affinity reagent and with the protein inhibitor cystatin B Subsequent analysis by flow cytometry shows that cysteine proteases are detectable on cytotoxic T lymphocytes by this approach, and that they increase upon TcR triggering. Evidence that they may be involved in self-protection comes from studies in which these cathepsin inhibitors induce apoptotic death in CTL within 4 hours when incubated on wells coated with antibodies cross-linking the T cell receptor but not control Mabs. These cathepsin inhibitors show negligible ability to kill resting CTL, and control reagents with the same reactive group but an inappropriate peptide fail to induce death after TcR engagement. This death of CTL is calcium-dependent, blocked by the granule toxin concanamycin B, and is not seen in perforin-deficient CTL. Human NK cells show a similar death in the presence of cathepsin inhibitors when incubated on wells coated with degranulating anti-CD16 Mab, but not control Mab. These results support the hypothesis that cytotoxic lymphocytes use proteolysis by surface cathepsins to protect themselves against membrane damage by secreted perforin. The nature of the cathepsin responsible for the above results is under investigation, and some evidence suggests both the lysosomal cathepsins B and L are expressed on the T lymphocyte surface. Cathepsin W is a novel cathepsin originally described as an espressed sequence tag and subsequently found in mRNA expression studies to be expressed exclusively in cytotoxic lymphocytes (both T and NK cells). Since it is an obvious candidate for the above activity, we have expressed human pre-procathepsin W in E. coli and made a series of monoclonal antibodies against it. In Western blots, some of these react with a single 30kd band in mouse CTL, a size expected for active cathepsin W. Experiments are currently under way to see if cathepsin W is localized in granules, on the cell surface, or elsewhere, and if it is involved in self-protection.